Methods, compositions and kits for detection and analysis of antibiotic-resistant bacteria

Abstract

The present invention relates generally to detection of antibiotic-resistant bacteria in a sample. In particular, the invention provides methods, compositions and kits for detecting and analyzing methicillin-resistant Staphylococcus aureus (MRSA) and other methicillin-resistant bacteria in a sample.

I claim: 
     
       1. A method of detecting methicillin-resistant  S. aureus  (MRSA) in a sample, said method comprising:
 (a) providing a first set of primers, wherein said first set of primers are complementary to at least a portion of a mecA polynucleotide sequence; 
 (b) providing a second set of primers, wherein said second set of primers are complementary to at least a portion of an MSSA-orfX polynucleotide sequence, wherein said MSSA-orfX polynucleotide sequence is from a region of the orfX gene surrounding the position at which an insertion cassette would be inserted in methicillin resistant  S. aureus  but is not inserted in methicillin susceptible  S. aureus;    
 (c) providing a third set of primers, wherein said third set of primers are complementary to at least a portion of an  S. aureus -specific polynucleotide sequence, and wherein said  S. aureus -specific polynucleotide sequence is not an orfX polynucleotide; 
 (d) combining said first, second and third set of primers with said sample in a reaction mixture; 
 (e) performing a multi-cycle amplification reaction with said reaction mixture; 
 (f) determining cycle numbers of appearance of each of said mecA, MSSA-orfX and  S. aureus -specific polynucleotide sequences,
 wherein said cycle numbers indicate whether MRSA is present in a sample. 
 
 
     
     
       2. A method according to  claim 1 , wherein if said  S. aureus -specific polynucleotide sequence appears at a cycle at least 4 cycles removed from appearance of said MSSA-orfX polynucleotide sequence, then said sample comprises both MRSA and methicillin-sensitive  S. aureus  (MSSA). 
     
     
       3. A method according to  claim 1 , wherein if said  S. aureus -specific polynucleotide sequence appears at a cycle within 3 cycles of said MSSA-orfX polynucleotide sequence, then said sample comprises MSSA and a non- S. aureus  methicillin resistant bacteria. 
     
     
       4. A method of identifying bacteria in a sample, said method comprising:
 (a) providing a first set of primers, wherein said first set of primers are complementary to at least a portion of a mecA polynucleotide sequence; 
 (b) providing a second set of primers, wherein said second set of primers are complementary to at least a portion of an MSSA-orfX polynucleotide sequence, wherein said MSSA-orfX polynucleotide sequence is from a region of the orfX gene surrounding the position at which an insertion cassette would be inserted in methicillin resistant  S. aureus  but is not inserted in methicillin susceptible  S. aureus;    
 (c) providing a third set of primers, wherein said third set of primers are complementary to at least a portion of an  S. aureus -specific polynucleotide sequence, and wherein said  S. aureus -specific polynucleotide sequence is not an orfX polynucleotide; 
 (d) combining said first, second and third set of primers with said sample in a reaction mixture; 
 (e) performing a multi-cycle amplification reaction with said reaction mixture; 
 (f) determining whether a mecA polynucleotide is present in said sample; 
 (g) determining whether an MSSA orfX polynucleotide is present in said sample; and 
 (h) determining whether an  S. aureus -specific polynucleotide is present in said sample, wherein said  S. aureus -specific polynucleotide is not an orfX polynucleotide,
 wherein the combination of (f), (g), and (h) present in said sample identifies bacteria in said sample. 
 
 
     
     
       5. A method according to  claim 4 , wherein if said mecA, polynucleotide and said  S. aureus -specific polynucleotide are both present in said sample and said MSSA-orfX polynucleotide is not present in said sample, then said sample comprises methicillin-resistant  S. aureus  (MRSA). 
     
     
       6. A method according to  claim 4 , wherein if said  S. aureus -specific polynucleotide and said MSSA-orfX are both present in said sample and said mecA polynucleotide is not present in said sample, then said sample comprises methicillin-susceptible  S. aureus  (MSSA) but does not comprise MRSA. 
     
     
       7. A method according to  claim 4 , wherein if said mecA polynucleotide and said MSSA-orfX polynucleotide are both present in said sample and said  S. aureus -specific polynucleotide is not present in said sample, then said sample comprises a non- S. aureus  methicillin-resistant bacteria and said sample does not comprise MRSA. 
     
     
       8. A method according to  claim 4 , wherein if only one of said mecA polynucleotide, said MSSA-orfX polynucleotide and said  S. aureus -specific polynucleotide are present in said sample, then said sample does not comprise MRSA. 
     
     
       9. A method according to  claim 4 , wherein said sample may comprise no bacteria, bacteria from a single strain, or a mixture of bacteria from more than one strain. 
     
     
       10. A method according to  claim 4 , Wherein said sample is a member selected from a bodily fluid, a nasal swab, and a tissue. 
     
     
       11. A method according to  claim 4 , wherein said determining whether a mecA polynucleotide is present in said sample, said determining whether an MSSA-orfX polynucleotide is present in said sample, and said determining whether an  S. aureus  specific polynucleotide is present in said sample are all conducted in separate aliquots of said sample. 
     
     
       12. A method according to  claim 4 , wherein two or more of said determining whether a mecA polynucleotide is present in said sample, said determining whether an MSSA-orfX polynucleotide is present in said sample, and said determining whether an  S. aureus -specific polynucleotide is present in said sample are conducted in identical aliquots of said sample. 
     
     
       13. A kit for identifying MRSA in a sample, said kit comprising:
 (a) a first set of primers complementary to at least a portion of a mecA polynucleotide sequence; 
 (b) a second set of printers complementary to at least a portion of MSSA-orfX polynucleotide sequence, wherein said MSSA-orfX polynucleotide sequence is from a region of the orfX gene surrounding the position at which an insertion cassette would be inserted in methicillin resistant  S. aureus  but is not inserted in methicillin susceptible  S. aureus;    
 (c) a third set of primers complementary to at least a portion of an  S. aureus -specific polynucleotide sequence, wherein said  S. aureus -specific polynucleotide sequence is not a bridging sequence; and 
 (d) at least one member selected from: a DNA polymerase enzyme, dNTPs, magnesium and a stabilizer.